Reorganization of the axonal membrane is often associated with pathological conditions that accompany demyelinating disorders such as multiple sclerosis. This research program focuses on the molecular organization and dynamics of the axonal membrane in developing, myelinating, and demyelinated nerve in tissue culture models. We will examine the distribution of excitable membrane components by immune-fluorescence and electron microscopy in myelinated fibers in the peripheral nervous systems and those fibers that have been experimentally demyelinated. Because we are particularly interested in the dynamic changes occurring in the organization of the axon membrane during myelination, we will monitor the sequential changes in excitable membrane protein topography by digital fluorescence microscopy on single neurons in long-term cultures of sensory neurons and Schwann cells as the axons become ensheathed. The role and magnitude of intracellular and/or extracellular elements that organize the axon membrane during development, ensheathment, and demyelination will be examined by measuring the lateral mobility of sodium channels, glycoproteins, and lipids by fluorescence photobleach recovery. Cultures of sensory neurons either in the absence or presence of Schwann cells will be used to assess the contribution of axon-glial contact in modulating the organization of the axon membrane during development. The role of the ensheathing cell in maintaining nodal and internodal specializations will be examined with developing, demyelinated, and remyelinating fibers. The temporal and spatial events in the expression, assembly, and segregation of NaChs with the extracellular matrix and cytoskeleton will be examined. Specifically, we will continue to study the role of both adhesion molecules, ankyrin, and spectrin in establishing restricted membrane domains that could serve to maintain excitable membrane components to specific regions of the axon.